NMDA mediates disruption of blood-brain barrier permeability via Rho/ROCK signaling pathway

Abstract

Glutamate can activate the N-methyl-D-aspartatic acid (NMDA) receptor (NMDAR), damage brain microvascular endothelial cells, and disturb the intercellular tight junctions (TJs). These result in changes in the permeability of the blood brain barrier (BBB). In neurons, the activation of Rho/ROCK signaling pathway is related to the activation of NMDAR，however, whether human brain vascular endothelial cells NMDAR mediates the Rho/ROCK pathway is not fully understood. The present study evaluates the effects of excessive NMDAR activation induced by NMDA (a glutamate analog) on the Rho/ROCK signaling pathway and the permeability of BBB by using a primary human brain microvascular endothelial cell (HBMEC) model. NMDAR subunit GluN1 was expressed in HBMECs and promoted by NMDA detected by Western blot and qRT-PCR. Furthermore, NMDA exposure decreased HBMEC viability, promoted HBMEC apoptosis, increased intracellular reactive oxygen species (ROS) levels, and destroyed the endothelial cytoskeleton. Additionally, NMDA exposure suppressed transendothelial electrical resistance (TEER) values and the expression of TJ proteins occludin and claudin5; it also promoted ROCK activated substrate myosin phosphatase target subunit-1 (MYPT)-1 phosphorylation and the transmittance of sodium fluorescein. In contrast, these effects were attenuated by ROCK inhibitor hydroxyfasudil (HF) and NMDAR antagonist MK801, respectively. Therefore, these results indicate that excessive endothelial NMDAR activation induced by NMDA may induce TJs and cytoskeleton damage, while HF attenuated NMDA-induced cytotoxicity in HBMECs by inhibiting the Rho/ROCK signaling pathway.